Most first-line chemotherapy drugs can destroy bulk tumor cells but fail to eliminate cancer stem cells, the cells that contribute to recurrence or relapse of the tumor, further progression, metastasis, and subsequent chemoresistance. This indicates that cancer stem cells may be “intrinsically” resistant to chemotherapy or that resistance is induced during first-line of therapy via acquisition of mutations, which are carried into and exist during the second-line of therapy settings. Therefore, the targeting of cancer stem cells as a first-line of therapy setting may eliminate intrinsically resistant cancer cells, prevent acquisition of resistance mutations, limit further progression and metastasis of cancer, and may also be applicable in the second-line therapy setting where responsive cancer cells can exist. Cancer cells, and more specifically cancer stem cells, can express one or multiple ATP Binding Cassette (ABC) transporters as a mechanism of resistance to chemotherapy drugs. ABC transporter proteins can facilitate the efflux of drugs from cancer cells rendering them resistant. The efflux of drugs from cancer cells means that higher concentrations of drug are required to achieve cell death, and at those concentrations the drug can be toxic to patients, essentially reducing the therapeutic index of the drug. Many known inhibitors of ABC transporters such as verapamil, reserpine, and cyclosporine, when used sequentially or in combination with other drugs, directly reduce or prevent the removal of the chemotherapeutic drug from the cell, making the drug more effective at lower concentrations. By increasing the intracellular concentration of the drug, and reducing initial treatment concentrations necessary to achieve cancer cell death the therapeutic window of the drug is improved and toxicity to the patient is alleviated. However, the concentration of ABC transport inhibitor necessary to turn off the transporters is too toxic to be used in patients, and the inhibitors are therefore not effective for use in combination therapy. Gottesman et al. (1993) Annu. Rev. Biochem. 62:385-427.
In some instances, ABC transporter activity is tightly regulated by sequestration of the transporter to intracellular compartments. Rocchi et al. (2000) Biochem. Biophys. Res. Commun. 271:42-6. For example, the translocation of ABC transporter, ABCG2, to the cell membrane is dependent on post-translational modification through phosphorylation by Akt kinase. Takada et al. (2005) Drug Metab. Dispos. 33:905-9. In cells expressing ABCG2, Akt inhibitors such as, Gleevec, LY294002, or LY335979, have been shown to reduce or completely eliminate translocation of transporter to the cell membrane and either reduce or completely abrogate transporter activity, thereby sensitizing resistant cells to drugs. Shepard et al. (2003) Int. J. Cancer 103:121-5; Nakanishi et al. (2006) Blood 108:678-84; Burger et al. (2005) Cancer Biol. Ther. 4:747-52; Ozvegy-Laczka et al. (2004) Mol. Pharmacol. 65:1485-95; Houghton et al. (2004) Cancer Res. 64:2333-7. However, it has been shown that this therapeutic strategy leads to compensatory elevations in transporter expression to maintain resistance, and is therefore insufficient for efficacious therapeutic applications.
Another strategy for overcoming ABC transporter-related drug resistance is to inhibit pathways that control ABC transporter expression in the resistant cancer cells, including cancer stem cells. The combination of Smo (smoothened) antagonist, cyclopamine, with chemotherapy drugs has been shown to reduce ABCG2 and ABCB1/MDR1 activity and to increase cancer cell death as compared to drug alone in vitro, by mechanisms that have yet to be identified. Singh et al. (2011) Oncogene 30:4874-86; Zhang et al. (2009) Neoplasia 11:96-101; Sims-Mourtada et al. (2007) Oncogene 26:5674-9; Lou et al. (2007) Oncogene 26:1357-60. However, cyclopamine is a toxic alkaloid that is lethal to humans with no feasible therapeutic application.
Itraconazole is a prescription-only antifungal agent that has been used to treat fungal infections such as, nail fungus, Aspergillosis, Candidiasis, Cryptococcosis, and Histoplasmosis. Hardin et al. (1988) Antimicrob. Agents Chemother. 32:1310-3. Itraconazole has also been shown to inhibit P-gp/MDR-1/ABCB1 activity directly. Miyama et al. (1998) Antimicrob. Agents Chemother. 42:1738-44. It has also been shown to be a strong CYP3A4, cytochrome P450 3A4 inhibitor. Tapaninen et al. (2011) J. Clin. Pharmacol. 51:359-67. Recently, itraconazole, arsenic trioxide, vitamin D3, and various other agents have been shown to inhibit the hedgehog pathway. Kim et al. (2010) Cancer Cell. 17:388-99. It was shown that these compounds could be used as single agents to inhibit growth or induce cell death of tumors containing a deregulated hedgehog pathway or mutations in Ptc, Smo or Gli proteins. Kim et al. (2013) Cancer Cell. 23:23-34. Itraconazole is currently in clinical trials for the treatment of several tumor types that are driven by the deregulation of the hedgehog pathway. Itraconazole has been shown to inhibit ABCG2 and ABCB1/MDR1 in cells that were artificially engineered to replicate acquired chemoresistance or in cells from heavily pretreated patients or patients treated as second-line of therapy in vitro. However, these experiments were performed using cytotoxic and non-therapeutic dosages in combination with dye substrates as a readout. Gupta et al. (1991) J. Clin. Invest. 87(4):1467-1469; Kurosawa et al. (1996) Ann. Hematol. 72(1):17-21. In the above context, acquired chemoresistance may be defined by when cancer cells are exposed to chemotherapeutic drugs until the cell “acquires” mutations that activate mechanisms and render the cancer cells resistant to chemotherapies.
Itraconazole has also been shown to increase survival of patients when administered in combination with second-line therapy for AML (acute myelogenous leukemia), ALL (acute lymphoblastic leukemia); Vreugdenhil et al. (1993) Ann. Hematol. 67(3):107-109, pancreatic cancer; Tsubamoto et al. (2015) Anticancer. Res. 35(7):4191-4196, biliary tract cancer; Tsubamoto et al. (2015) Anticancer. Res. 35(9):4923-4927, triple-negative breast cancer; Tsubamoto et al. (2014) Anticancer. Res. 34(7):3839-3844, ovarian cancer; Tsubamoto et al. (2014) Anticancer. Res. 34(5):2481-2487, and non-squamous NSCLC (non-small cell lung carcinoma) Rudin et al. (2013) J. Thorac. Oncol. 8(5):619-623. However, in these contexts itraconazole was administered to heavily pretreated patients in the second-line of therapy settings. Those patients may have acquired mutations conferring resistance to the chemotherapies due to their prior treatment with the chemotherapeutic agents.
There is thus a need for improved compounds, compositions, packaged pharmaceuticals, and methods for overcoming chemoresistance in tumor cells, particularly in tumor cells expressing ABC transporters, as first-line of therapy.